There is active implementation of molecular imaging in which living cells are targeted and visualized and imaged or imaging is performed to visualize targeted molecules in a living body, thereby clarifying molecular kinetics, intermolecular interaction and molecular position information, intending leading to elucidation of mechanism of life science and screening of new drugs. In particular, there are also active studies for detecting cancer cells and cancer lesions by visualizing abnormal cells, for example, cancer cells.
Most of six-carbon sugars (hexose) represented by glucose (grape sugar), for example, glucose, fructose, galactose and mannose play a critical role in activity of living organisms. Especially, glucose is known as the most important energy source for supporting cell lives in living things from mammals to Escherichia coli and yeast, and in particular, brain uses glucose as the sole energy source. Glucose includes mirror isomers: D-glucose and L-glucose, and only D-glucose among them can be utilized as an energy source by living organisms, and a living cell has a mechanism for taking up D-glucose selectively via transporter proteins in plasma membrane, such as glucose transporters and the like, and utilizing in the cell.
The six-carbon sugar (hexose), of which D-form occurs abundantly in nature and L-form as its optical isomer does not, or scarcely occurs, includes D-galactose, D-fructose and D-mannose in addition to glucose.
D-galactose is a sugar utilized as an energy source, contained abundantly in milk, fruits and vegetables, and additionally, produced at a rate of about 2 g per day also in a human body. For example, disaccharide lactose occupying 2 to 8% of milk is formed by D-galactose and D-glucose via glycoside linkage, and it is known that both the constituents are separated by lactase in absorption into small intestine, and absorbed into a body via SGLT a sort of glucose transporter. When D-galactose is transported from small intestinal epithelial cells into blood vessels, it passes through a glucose transporter GLUT2. Galactose taken up into cells undergoes phosphorylation at 1-position, then, enters the glycolytic pathway and is utilized as energy, or utilized for biosynthesis of glycolipid and glycoprotein. On the other hand, L-galactose is described as an intermediate metabolite in the Smirnoff-Wheeler pathway which is one of pathways when an antioxidant substance vitamin C (L-ascorbic acid) which cannot be biosynthesized by primates is biosynthesized from D-glucose in a plant, but is a rare sugar which is not usually seen in biology in general.
2-deoxy-2[18F]fluoro-D-galactose obtained by labeling D-galactose with 18F has an example of application for analyzing metabolites in liver (non-patent document 1). 2-deoxy-2[18F]fluoro-D-galactose has been reported to have a possibility of utilization for imaging of galactose metabolism in cancer, it has not been generalized, though (non-patent document 2).
D-fructose is also called fruit sugar, and is contained in large amounts in berries and fruits such as melon and the like and some kinds of root vegetables, produced also in the body, in addition. Ingested D-fructose is taken up into epithelial cells via a glucose transporter GLUT5 in small intestinal epithelium, then, enters mainly through GLUT2 into blood. Fructose, which has entered into hepatic cells, undergoes phosphorylation by fructokinase, and is used for synthesis of fatty acids and energy production, and in addition, converted also into D-glucose. Since GLUT5 is expressed also in smooth muscle, kidney, adipocyte, brain and testis, it is thought that GLUT5 plays important functions in these regions respectively, and for example, D-fructose is used as an energy source in sperm motility as well. Among the corn syrup that is widely circulated as a food sweetener, those having increased content of D-fructose, which is cheap and shows intense sweetness particularly at low temperatures, are used in large amounts in refreshing beverages and the like, and excessive intake of D-fructose exerts a bad influence on neuronal activity in brain and is considered dangerous as a trigger of obesity and cancers. There is a paper reporting that L-fructose can be utilized to some extent when eaten, but it has been also speculated that this may be due to a conversion by enterobacteria.
1-deoxy-1-[18F]fluoro-D-fructose has been synthesized as a radiolabeled compound and moderate uptake thereof into tumor has been reported, however, this molecule appears to undergo no metabolism in a cell, and therefore, is not used. Recently, 6-deoxy-6-[18F]fluoro-D-fructose, which is metabolized intracellularly, has been synthesized and reported as a candidate tracer for PET targeting uptake thereof via GLUT5 in breast cancer (non-patent document 3).
D-mannose is contained in fruits and fruit peel and the like. A polysaccharide composed mainly of mannose is called mannan, and contained in plants, yeasts and bacteria. Konjac contains as the main component glucomannan composed of mannose and glucose. D-mannose is, when orally taken in case of human, believed to be mostly excreted into urine in the usual case, and the way of uptake thereof in a human body is unclear in many aspects. When taken into a cell, D-mannose is phosphorylated, then, converted into fructose 6-phosphate, which is an intermediate in the glycolytic pathway.
A mannose receptor to which D-mannose binds specifically is helpful for eliminating high mannose glycoprotein, which increases during inflammation. For example, there is a high mannose sugar chain region on the membrane surface of P. carini, which is a causative microorganism of carinii pneumonia, a kind of opportunistic infection occupying the first cause of AIDS patients' death, and a mannose receptor occurring on alveolar macrophage recognizes this, thereby promoting migration of macrophage. Not only D-mannose but also L-galactose has a strong macrophage stimulating action, and additionally, both D-mannose and L-galactose are used as a precursor for biosynthesis of vitamin C in plants.
Though it is reported that [18F]-2-fluoro-2-deoxy-D-mannose can be used as a cancer tracer, but this is not popularized (non-patent document 4, non-patent document 5).
As described above, various hexoses such as represented by glucose play an important role in living organisms. However, all studies to examine the relationship between these hexoses and cells have a common issue as described below taking D-glucose as a typical example.
Conventionally, studies on how living organisms take up D-glucose into cells and utilize it have been conducted, for example, by measuring the intracellular quantity of a radio isotope using D-glucose labeled with the radio isotope or its derivatives (D-deoxyglucose or the like). This method is excellent for quantification, however, has a problem of low sensitivity, and in addition, it has a defect that D-glucose uptake into living cells cannot be observed continuously in real time due to the methodology of measurement. Then, the group of the present inventors has proposed a method of using green fluorescence emitting 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) obtained by linking an N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino group as a fluorescent chromophore at the 2-position of D-deoxyglucose, as a method which can be used in a study of the dynamic process of D-glucose uptake into living cells, and has demonstrated its usefulness using various cells of mammals (non-patent document 6).
This method uses a property of 2-NBDG which is selectively taken up into living cells, and since the dynamic activity of D-glucose uptake into a cell can be observed in a quantitative manner by tracing the change in the fluorescence intensity due to the uptake, this method is evaluated by researchers around the world as a ground-breaking method for studying how a living organism takes up D-glucose into a cell and utilizes it, and now, regarded as a standard protocol essential in this study field (non-patent document 7). Further, for evaluating specific uptake of D-glucose, the group of the present inventors has developed green fluorescence emitting 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG) obtained by linking an N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino group as a fluorescent chromophore at the 2-position of L-deoxyglucose, the enantiomer of D-deoxyglucose, and has also developed a L-deoxyglucose which is a glucose derivative emitting red fluorescence color (2-TRLG) in which sulforhodamine 101 is bound at its 2-position via sulfonamide-linking (patent document 1).
Further, there is a report of application of a molecule (1-NBDF), in which NBD is linked to the 1-position of D-fructose, to breast cancer (non-patent document 8).
As such, glucose derivatives and fructose derivatives bearing NBD in the molecule are known as fluorescently labeled sugar derivatives capable of imaging living cells at the cellular level individually.
In addition, a fluorescent glucose derivative obtained by linking a blue fluorescence emitting coumarin derivative molecule to D-glucose is known as well (Esculin, Fraxin, patent document 2). However, since there is no report of using a sugar derivative bearing a blue fluorescent molecule for imaging living cells at the cellular level individually, a blue fluorescence-labeled sugar derivative which can be used for imaging at the cellular level has been long-awaited.
It is known that tumor cells showing active proliferation potential require glucose as their energy source and material source for their synthesis of amino acids, nucleic acids, lipids and the like more than usual cells. Utilizing this property, a technique to diagnose cancer non-invasively from the outside of the body has already been put to practical use in the clinical medicine field, wherein 18F-radiolabeled D-glucose derivative 18F-fluoro-2-deoxy-D-glucose (FDG) is administered to a patient, and gamma ray radiated by 18F decay in FDG, taken up into tumor tissue and accumulated in the cell, is detected by a PET (positron emission tomography) apparatus. The PET examination using this radiolabeled D-glucose derivative has a issue of inability to detect micro cancer having a potential of rapid growth due to lack in spatial resolution capable of discriminating individual cells (the lower limit of spatial resolution is practically about 5 mm in PET examination). FDG faces the challenges of its short half-life (110 minutes) and the need for large-scale facilities, in addition. Further, the radiolabeled FDG which is a D-glucose derivative has a big challenge of how to avoid the fundamental problem of uptake thereof not only into tumor cells but also into normal tissue and normal cells. Particularly since adipose tissue and muscle distributing throughout the whole body, small intestinal epithelium, liver and the like take up D-glucose so strongly, discriminating them from tumor is problematic.
Other hexoses have also been tried to be applied to detect and image cancer by using their radio-labeled compounds as described above. Like D-glucose, however, its use is limited due to D-configuration thereof, and additionally, there is a problem of inability to detect a difference in individual single cells in real time with accuracy.
Application of a fluorescently labeled D-glucose derivative to tumor imaging is now underway actively in various countries intending to improve spatial resolution which is a weak point of a radiolabeling method, simultaneously avoiding the complication and danger of radiolabeling, and enabling instantaneous detection with a simple apparatus. 2-NBDG as a fluorescently labeled D-glucose derivative is one of typical molecules thereof, and it has been reported that 2-NBDG is well taken up into a tumor cell as FDG is (non-patent document 9, patent document 3, and the like), and there are trials of applying 2-NBDG to cancer diagnostic imaging (non-patent document 10, non-patent document 11).
There are active trials linking to D-glucose a fluorescent molecule emitting fluorescence of which wavelength longer than 2-NBDG such as red or near-infrared region showing higher tissue-penetrability and brighter fluorescence than 2-NBDG, for enabling fluorescence detection even from deeper tissue as compared with the case when 2-NBDG is used (non-patent document 12, non-patent document 13, non-patent document 14, and the like). However, since all of these novel fluorescent molecules have molecular weights and sizes much larger than NBD, any of fluorescent glucose derivatives to which these have been linked cannot pass through a glucose transporter (GLUT).
All fluorescent glucose derivatives so far reported including 2-NBDG are fluorescent derivatives containing D-(+)-glucose as a scaffold, and have the fundamental problem of being taken up into normal cells as well like radiolabeled FDG.
On the other hand, an idea of discriminating cancer by an approach utilizing the result of metabolic activity of cancer cells is proposed and attracting notice (non-patent document 15). A cancer cell showing brisk metabolic activity generates a large amount of acids in the form of CO2 and proton (H+) in the cell due to metabolism. Such acids corresponding to wastes, so to speak, are eliminated or neutralized in normal cells' case with the aid of the circulation system such as blood flow and the like, to prevent acidification in the cell. However, tissue, which is constructed to match the metabolic activity of normal cells, cannot cope with cancer cells continuing unexpected growing activity. Especially within cancer tissue remote from blood vessels, elimination and neutralization of acids tend to be insufficient, and cancer cells try to prevent intracellular acidification by developing various molecular mechanisms. A strategy targeting such a molecule particularly advanced in cancer cells might be useful for developing, for example, diagnostic pharmaceuticals which selectively discriminate cancer cells in hypoxic condition (these are known as cancer cells resistant to radiation and drugs) and a drug delivery system for carrying anti-cancer agents. As one of such target molecules, the carbonic anhydrase group expressing excessively on the plasma membrane of a cancer cell has been attracting attention (non-patent document 15).
Excess CO2 as an acidic waste inevitably generated in a cell in the body by the cellular metabolic activity is eliminated by various in vivo mechanisms, to prevent acidification in the cell. A key supporting these processes is elimination of an acid by blood flow. However, in the case of cancer cells located in solid cancer dozens of microns or more away from blood vessels or abnormally growing cells in the position facing the inner cavity of a digestive tract and far from blood vessels, oxygen and glucose supply is lacking and elimination of acids as metabolites tends to be insufficient. It has recently been reported that some of such cancer cells carrying out metabolism in hypoxic and low-nutrition environment support elimination of CO2 from the inside of a cell and neutralization of acids generated in a cell, by excessively expressing membrane-spanning carbonic anhydrases (CA 9 and CA 12) in the plasma membrane (non-patent document 15). Supuran and colleagues have found that a derivative of fluorescent low molecular weight compound coumarin binds to carbonic anhydrases (for example, CA 9 is supposed) expressing strongly on the plasma membrane of some cancer cells under hypoxic condition, to inhibit decarboxylating action of these enzymes (non-patent document 16, patent document 2). These coumarin derivatives are expected as one of candidates of the next generation anti-cancer agents for the reason that the derivatives attack cancer cells by destructing the pH balance of the cancer cells under the hypoxic condition (non-patent document 21).
However, carbonic anhydrases are enzymes essential for the life of all cells, and in mammals, 16 kinds of isozymes are present not only on the surface of plasma membrane but also in cytoplasm and mitochondria. Therefore, it is required that the above-described fluorescent low molecular weight compound does not cause side effect by inhibiting other types of carbonic anhydrases present in normal cells. One effective strategy is that fluorescent low molecular weight compounds such as coumarin derivatives and the like act selectively on CA9 or the like having the reaction site on the outside of the plasma membrane of a cancer cell, to prevent invasion into the cell. For this purpose, an idea is suggested in which a charge is introduced into a compound or a glycoside is prepared to give hydrophilicity to the molecule, thereby preventing penetration through plasma membrane constituted of lipid bilayer membranes (non-patent document 17). For example, Supuran and colleagues suggest that various coumarins or derivatives thereof are linked to the 1-position of a natural sugar such as D-glucose, D-mannose, D-galactose, L-rhamnose and the like, to give water solubility to the molecule, thereby providing plasma membrane impermeability (patent document 2). However, the 1-position is easily subjected to hydrolysis, and when a natural sugar is used, an influence on normal cells cannot be avoided.
In recent years, as a method for utilizing molecules showing increased expression in tumor cells, fluorescent molecular markers obtained by linking a fluorescent molecule to a molecule other than glucose are under active development. Examples thereof include those utilizing the RGD sequence and those utilizing EGF, and the like (non-patent document 18). However, such methods have a problem analogous to the method of using a derivative of a natural sugar (for example, D-glucose) as well, since even in such methods fluorescent molecules are basically taken up into normal cells though there is a difference of the degree of uptake. In contrast, a molecular marker targeting a specific tumor cell using a specific antibody or the like cannot determine other types of tumors, thus, versatility thereof is problematic.